Glycosylceramides perform important immunological functions as T cell antigens presented by CD1, a family of lipid-binding MHC-like molecules. For example, alphaGalactosylCeramides (alphaGC) specifically activate regulatory NKT cells which modulate, through the release of either Th1 or Th2 cytokines, various disease conditions, and other glycosylceramides may elicit adaptive T cell responses involved in Multiple Sclerosis. Thus, the program project uses glycosylceramides as a prime model antigen family to explore fundamental issues of glycolipid antigen presentation by CD1 molecules with potential clinical applications. The project brings together several new tools and methodologies developed in a multidisciplinary collaboration. Dr. Savage (Project #1) is generating an extended set of synthetic glycolipids modeled around alphaGC whose physical and structural interactions with CD1d and TCR are studied by Dr. Teyton (Project #2), and which we will use in this proposal to systematically explore, in the mouse system, antigen presentation by CD1d and the corresponding TCR repertoire and T cell functions. The specific aims are 1) To study T cell recognition of glycosylceramides in vivo using CD1d tetramer technology, and compare their adjuvant effects; 2) To study the Th1 vs Th2 modulatory properties of selected alphaGC variants in IDDM, EAE and rejection of liver metastasis; 3) To study how glycosylceramide structure dictates intracellular trafficking and access to endosomal compartments, using fluorochrome and radio-labeled glycolipids and developing new methodologies for the detection and imaging of CD1d-alphaGC complexes. Because glycolipids do not mutate and CD1 molecules are highly conserved, these basic studies in mice lay the foundations for the development of universal lipid-based vaccines as well as the rational design of glycosylceramide adjuvants which can modulate autoimmunity, cancer and infections.